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Summary: Genetic engineering of flower color provides biotechnological products such as blue carnations or roses by accumulating delphinidin‐based anthocyanins not naturally existing in these plant species. Betalains are another class of pigments that in plants are only synthesized in the order Caryophyllales. Although they have been engineered in several plant species, especially red‐violet betacyanins, the yellow betaxanthins have yet to be engineered in ornamental plants.We attempted to produce yellow‐flowered gentians by genetic engineering of betaxanthin pigments. First, white‐flowered gentian lines were produced by knocking out the dihydroflavonol 4‐reductase (DFR) gene using CRISPR/Cas9‐mediated genome editing.Beta vulgaris BvCYP76AD6 and Mirabilis jalapa MjDOD, driven by gentian petal‐specific promoters, flavonoid 3′,5′‐hydroxylase (F3′5′H) and anthocyanin 5,3′‐aromatic acyltransferase (AT), respectively, were transformed into the above DFR‐knockout white‐flowered line; the resultant gentian plants had vivid yellow flowers. Expression analysis and pigment analysis revealed petal‐specific expression and accumulation of seven known betaxanthins in their petals to c. 0.06–0.08 μmol g FW−1.Genetic engineering of vivid yellow‐flowered plants can be achieved by combining genome editing and a suitable expression of betaxanthin‐biosynthetic genes in ornamental plants. [ABSTRACT FROM AUTHOR]

The lacquer tree, Toxicodendron vernicifluum, is a common industrial crop in East Asia. However, T. vernicifluum seeds are extremely difficult to germinate, which poses a major obstacle to establishing seedlings for sap production. In this study, we examined the germination properties of T. vernicifluum seeds in order to establish an inexpensive and effective method to promote seed germination. The seeds are covered with a hard endocarp, which we degrade using conventional sulfuric acid-based methods. Although sulfuric acid was effective in promoting seed germination, the germination rate was less than 5%. In addition to treatment with sulfuric acid, co-treatment with cold temperatures or the phytohormone gibberellic acid increased the germination rate to 22–35%. Seed viability analysis combined with specific gravity-based seed selection revealed that more than half of the seeds housed embryos that were incapable of germination. In additions, specific gravity-based seed selection aided in the selection of seeds capable of germination and improved the germination rate to approximately 47%. Taken together, our results suggest that the low germination rate of T. vernicifluum seeds is due to deep seed dormancy—which is controlled by physical and physiological mechanisms—and low embryo viability. To improve the germination rate of T. vernicifluum seeds, we propose an effective method whereby seeds with good germination capacity are selected based on specific gravity, following which their physiological dormancy is inactivated through cold pretreatment. [ABSTRACT FROM AUTHOR]

Gentians cultivated in Japan (Gentiana triflora and Gentiana scabra and hybrids) have blue flowers, but flower colour intensity differs among cultivars. The molecular mechanism underlying the variation in flower colour intensity is unclear. Here, we produced F2 progeny derived from an F1 cross of intense- and faint-blue lines and attempted to identify the genes responsible for flower colour intensity using RNA-sequencing analyses. Comparative analysis of flower colour intensity and transcriptome data revealed differentially expressed genes (DEGs), although known flavonoid biosynthesis-related genes showed similar expression patterns. From quantitative RT-PCR (qRT-PCR) analysis, we identified two and four genes with significantly different expression levels in the intense- and faint-blue flower lines, respectively. We conducted further analyses on one of the DEGs, termed GtMIF1 , which encodes a putative mini zinc-finger protein homolog, which was most differently expressed in faint-blue individuals. Functional analysis of GtMIF1 was performed by producing stable tobacco transformants. GtMIF1 -overexpressing tobacco plants showed reduced flower colour intensity compared with untransformed control plants. DNA-marker analysis also confirmed that the GtMIF1 allele of the faint-blue flower line correlated well with faint flower colour in F2 progeny. These results suggest that GtMIF1 is one of the key genes involved in determining the flower colour intensity of gentian. [ABSTRACT FROM AUTHOR]

Summary: Cultivated Japanese gentians traditionally produce vivid blue flowers because of the accumulation of delphinidin‐based polyacylated anthocyanins. However, recent breeding programs developed several red‐flowered cultivars, but the underlying mechanism for this red coloration was unknown. Thus, we characterized the pigments responsible for the red coloration in these cultivars. A high‐performance liquid chromatography with photodiode array analysis revealed the presence of phenolic compounds, including flavones and xanthones, as well as the accumulation of colored cyanidin‐based anthocyanins. The chemical structures of two xanthone compounds contributing to the coloration of red‐flowered gentian petals were determined by mass spectrometry and nuclear magnetic resonance spectroscopy. The compounds were identified as norathyriol 6‐O‐glucoside (i.e., tripteroside designated as Xt1) and a previously unreported norathyriol‐6‐O‐(6′‐O‐malonyl)‐glucoside (designated Xt2). The copigmentation effects of these compounds on cyanidin 3‐O‐glucoside were detected in vitro. Additionally, an RNA sequencing analysis was performed to identify the cDNAs encoding the enzymes involved in the biosynthesis of these xanthones. Recombinant proteins encoded by the candidate genes were produced in a wheat germ cell‐free protein expression system and assayed. We determined that a UDP‐glucose‐dependent glucosyltransferase (StrGT9) catalyzes the transfer of a glucose moiety to norathyriol, a xanthone aglycone, to produce Xt1, which is converted to Xt2 by a malonyltransferase (StrAT2). An analysis of the progeny lines suggested that the accumulation of Xt2 contributes to the vivid red coloration of gentian flowers. Our data indicate that StrGT9 and StrAT2 help mediate xanthone biosynthesis and contribute to the coloration of red‐flowered gentians via copigmentation effects. [ABSTRACT FROM AUTHOR]

Abstract: The objective of the present study was to evaluate the effectiveness of enrofloxacin (ERFX) as a second‐line antibiotic for treatment of acute Escherichia coli (E. coli) mastitis. Forty‐two cows with naturally occurring acute E. coli mastitis were enrolled. On the first day of treatment (day 0), empirically selected antibiotics (oxytetracycline: n = 32, kanamycin: n = 10) were administered. Although systemic signs improved in 10 cows (first‐line group), the signs remained unchanged or worsened in 32 cows on day 1, including two cows that were found dead. The 30 surviving cows were randomly assigned to second‐line groups constituting an ERFX group (n = 19) or a control group (n = 11) that was treated with other antibiotics. Response to each treatment was evaluated by measuring clinical signs from day 0 to day 3, subsequent quarter milk recovery, and the 60‐day survival rate. Appetite on day 3 was significantly better in the ERFX group compared to the control group. No significant differences were observed in the 60‐day survival rate or the subsequent milk recovery between the ERFX group and the control group. Thus, the use of ERFX as a second‐line antibiotic for the treatment of acute E. coli mastitis could induce a rapid appetite recovery. [ABSTRACT FROM AUTHOR]

The disinfection effect of slightly acidic electrolyzed water ( SAEW) use in a farm where Pseudomonas mastitis has spread was evaluated. Despite the application of antibiotic therapy and complete cessation of milking infected quarters, numerous new and recurrent Pseudomonas aeruginosa clinical mastitis infections (5.8-7.1% of clinical mastitis cases) occurred on the farm from 2003 to 2005. Procedural changes and equipment modifications did not improve environmental contamination or the incidence of Pseudomonas mastitis. To more thoroughly decontaminate the milking parlor, an SAEW system was installed in 2006. All milking equipment and the parlor environment were sterilized with SAEW ( pH 5-6.5, available chlorine 12 parts per million) before and during milking time. After adopting the SAEW system, the incidence of clinical and subclinical Pseudomonas mastitis cases decreased significantly ( P < 0.0001) and disappeared. These findings suggest that SAEW effectively reduced the incidence of mastitis in a herd contaminated by Pseudomonas species. This is the first report to demonstrate the effectiveness of disinfection by SAEW against mastitis pathogens in the environment. [ABSTRACT FROM AUTHOR]

Exercise intolerance and claudication are symptomatic of peripheral arterial disease. There is a close relationship between muscle O2 delivery, microvascular oxygen partial pressure (PmvO2), and contractile performance. We therefore hypothesized that a reduction of hemoglobin-oxygen affinity via RSR13 would maintain a higher PmvO2 and enhance blood-muscle O2 transport and contractile function. In male Wistar rats (12 wk of age), we created hindlimb ischemia via right-side iliac artery ligation (AL). The contralateral (left) muscle served as control (CONT). Seven days after AL, phosphorescence-quenching techniques were used to measure PmvO2 at rest and during contractions (electrical stimulation; 1 Hz, 300 s) in tibialis anterior muscle (TA) under saline (n = 10) or RSR13 (n = 10) conditions. RSR13 at rest increased TA PmvO2 in CONT (13.9 ± 1.6 to 19.3 ± 1.9 Torr, P < 0.05) and AL (9.0 ± 0.5 to 9.9 ± 0.7 Torr, P < 0.05). Furthermore, RSR13 extended maintenance of the initial TA force (i.e., improved contractile performance) such that force was not decreased significantly until contraction 240 vs. 150 in CONT and 80 vs. 20 in AL. This improved muscle endurance with RSR13 was accompanied by a greater ΔPmvO2 (PmvO2 decrease from baseline) (CONT, 7.4 ± 1.0 to 11.2 ± 1.3; AL, 6.9 ± 0.5 to 8.6 ± 0.6 Torr, both P < 0.05). Whereas RSR13 did not alter the kinetics profile of PmvO2 (i.e., mean response time) substantially during contractions, muscle force was elevated, and the ratio of muscle force to PmvO2 increased. In conclusion, reduction of hemoglobin-oxygen affinity via RSR13 in AL increased PmvO² and improved muscle contractile performance most likely via enhanced blood-muscle O2 diffusion. NEW & NOTEWORTHY This is the first investigation to examine the effect of RSR13 (erythrocyte allosteric effector) on skeletal muscle microvascular oxygen partial pressure kinetics and contractile function using an arterial ligation model of peripheral arterial disease in experimental animals. The present results provide strong support for the concept that reducing hemoglobin-O2 affinity via RSR13 improved tibialis anterior muscle contractile performance most likely via enhanced blood-muscle O2 diffusion. [ABSTRACT FROM AUTHOR]

Milking performance of milking machines that matches the production capability of dairy cows is important in reducing the risk of mastitis, particularly in high-producing cows. This study used a simulated milking device to examine the milking performance of the milking system of 73 dairy farms and to analyze the factors affecting claw vacuum. Mean claw vacuum and range of fluctuation of claw vacuum (claw vacuum range) were measured at three different flow rates: 5.7, 7.6 and 8.7 kg/min. At the highest flow rate, only 16 farms (21.9%) met both standards of mean claw vacuum ≥35 kPa and claw vacuum range ≤ 7 kPa, showing that milking systems currently have poor milking performance. The factors affecting mean claw vacuum were claw type, milk-meter and vacuum shut-off device; the factor affecting claw vacuum range was claw type. Examination of the milking performance of the milking system using a simulated milking device allows an examination of the performance that can cope with high producing cows, indicating the possibility of reducing the risk of mastitis caused by inappropriate claw vacuum. [ABSTRACT FROM AUTHOR]

Dendritic phosphors were obtained by the stepwise integration of BiCl3 in phenylazomethine dendrimers. The bismuth-coordinated phenylazomethines displayed photoluminescence at 500-800 nm, and the intensity could be tuned by changing the stoichiometry of BiCl3 and the dendrimer. This phosphor did not show serious luminescence quenching even though the local concentration of BiCl3 in the dendrimer was as high as 20 M, and luminescence was also observed in the solid state. The absorption and emission properties could be reversibly switched by addition of a Lewis base or under electrochemical redox control, which induced the reversible complexation of BiCl3 in the dendrimer. [ABSTRACT FROM AUTHOR]

Parrots and songbirds learn their vocalizations from a conspecific tutor, much like human infants acquire spoken language. Parrots can learn human words and it has been suggested that they can use them to communicate with humans. The caudomedial pallium in the parrot brain is homologous with that of songbirds, and analogous to the human auditory association cortex, involved in speech processing. Here we investigated neuronal activation, measured as expression of the protein product of the immediate early gene ZENK, in relation to auditory learning in the budgerigar (Melopsittacus undulatus), a parrot. Budgerigar males successfully learned to discriminate two Japanese words spoken by another male conspecific. Re-exposure to the two discriminanda led to increased neuronal activation in the caudomedial pallium, but not in the hippocampus, compared to untrained birds that were exposed to the same words, or were not exposed to words. Neuronal activation in the caudomedial pallium of the experimental birds was correlated significantly and positively with the percentage of correct responses in the discrimination task. These results suggest that in a parrot, the caudomedial pallium is involved in auditory learning. Thus, in parrots, songbirds and humans, analogous brain regions may contain the neural substrate for auditory learning and memory. [ABSTRACT FROM AUTHOR]

The physical characteristics of vocalization were examined in budgerigars ( Melopsittacus undulatus) deafened at the age of about 28 days. Autocorrelation was used in the analysis, with 2 parameters: 1) inter-element intervals and 2) amplitudes of all sound elements of the vocalization. The deafened birds developed the temporal pattern specific to normal warble song in autocorrelograms. However, abnormalities were found in the frequency spectrum of elements. The temporal pattern of warble song might be an innate character, because auditory feedback is unnecessary for its development. [ABSTRACT FROM AUTHOR]

Background: CRISPR/Cas9 technology is one of the most powerful and useful tools for genome editing in various living organisms. In higher plants, the system has been widely exploited not only for basic research, such as gene functional analysis, but also for applied research such as crop breeding. Although the CRISPR/Cas9 system has been used to induce mutations in genes involved in various plant developmental processes, few studies have been performed to modify the color of ornamental flowers. We therefore attempted to use this system to modify flower color in the model plant torenia (Torenia fournieri L.). Results: We attempted to induce mutations in the torenia flavanone 3-hydroxylase (F3H) gene, which encodes a key enzyme involved in flavonoid biosynthesis. Application of the CRISPR/Cas9 system successfully generated pale blue (almost white) flowers at a high frequency (ca. 80% of regenerated lines) in transgenic torenia T0 plants. Sequence analysis of PCR amplicons by Sanger and next-generation sequencing revealed the occurrence of mutations such as base substitutions and insertions/deletions in the F3H target sequence, thus indicating that the obtained phenotype was induced by the targeted mutagenesis of the endogenous F3H gene. Conclusions: These results clearly demonstrate that flower color modification by genome editing with the CRISPR/Cas9 system is easily and efficiently achievable. Our findings further indicate that this system may be useful for future research on flower pigmentation and/or functional analyses of additional genes in torenia. [ABSTRACT FROM AUTHOR]

Examines the effect of hydrostatic pressure on the internal rotation rate of macrocyclic compounds using the DNMR method. Use of quartz cells for high-pressure experiment analysis; Acceleration of the benzene ring rotation; Shrinkage of the methylene bridge structure upon pressurization.

Objective: To establish a threshold value of bovine leukemia virus (BLV) proviral load (PVL) to identify increased risk of severe clinical mastitis, and to examine the prognosis and economic loss of clinical mastitis based on the newly established PVL cut-off value., Animals: 97 lactating Holstein cows with clinical mastitis., Methods: Blood and milk samples were collected aseptically from each cow. Youden index was used for receiver-operating characteristic curve analysis with the severity rate of clinical mastitis as the dependent variable and PVL as an independent variable. PVL cut-off value was used as a criterion to compare the severity rate of clinical mastitis, percentage of cows with and without systemic treatments, number of treatments, cost of treatment, and prognosis., Results: PVL cut-off value was 17.8 copies/10 ng DNA for the dependent variable MILD vs SEVERE. The severity rate of clinical mastitis, percentage of cows given systemic treatments, and technical fees for medical treatment were significantly higher in the group above the PVL cut-off value than in the group below the PVL cut-off value and the negative group. Number of treatments was significantly higher in the group above the cut-off value than in the group below the cut-off value. There was no significant difference in prognosis after mastitis among the 3 groups., Clinical Relevance: These results suggested that PVL cut-off value of 17.8 copies/10 ng DNA was a useful threshold for increased economic losses in BLV-infected cows; it may also serve as a new standard value for the detection and culling of BLV-infected cows in Japan.

Japanese cultivated gentians are highly valued ornamental flowers in Japan, but the flower shape is mostly limited to the single-flower type, unlike other flowers such as roses and carnations. To overcome this limitation, we used the CRISPR/Cas9 genome editing system to increase double-flowered genetic resources in gentians. Our approach targeted an AGAMOUS ( AG ) floral homeotic gene ( AG1 ), which is responsible for the natural mutation that causes double flowers in gentians. We designed two targets in exon 1 of AG1 for genome editing and found that 9 of 12 herbicide-resistant shoots had biallelic mutations in the target regions of AG1 . These nine lines all produced double flowers, with stamens converted into petaloid organs, similar to the natural mutant. We also analyzed the off-target effects of AG2 , which is homologous to AG1 , and found that such effects occurred in gentian genome editing but with low frequency. Furthermore, we successfully produced transgene-free genome-edited plants (null segregants) by crossing with wild-type pollen. F 1 seedlings were subjected to PCR analysis to determine whether foreign DNA sequences, two partial regions of the CaMV35S promoter and Cas9 gene, were present in the genome. As a result, foreign genes were segregated at a 1 : 1 ratio, indicating successful null segregant production. Using PCR analysis, we confirmed that four representative null segregants did not contain transfer DNA. In summary, our study demonstrates that the CRISPR/Cas9 system can efficiently produce double-flowered gentians, and null segregants can also be obtained. These genome-edited plants are valuable genetic resources for future gentian breeding programs., Competing Interests: Conflict of interestThe authors declare no conflict of interest., (© 2023 Japanese Society for Plant Biotechnology.)

Flower opening is important for successful pollination in many plant species, and some species repeatedly open and close their flowers. This is thought to be due to turgor pressure changes caused by water influx/efflux, which depends on osmotic oscillations in the cells. In some ornamental plants, water-transporting aquaporins, also known as plasma membrane intrinsic proteins (PIPs), may play an important role in flower opening. However, the molecular mechanism(s) involved in corolla movement are largely unknown. Gentian (Gentiana spp.) flowers undergo reversible movement in response to temperature and light stimuli; using gentian as a model, we showed that the Gentiana scabra aquaporins GsPIP2;2 and GsPIP2;7 regulate repeated flower opening. In particular, phosphorylation of a C-terminal serine residue of GsPIP2;2 is important for its transport activity and relates closely to the flower re-opening rate. Furthermore, GsPIP2;2 is phosphorylated and activated by the calcium (Ca2+)-dependent protein kinase GsCPK16, which is activated by elevated cytosolic Ca2+ levels in response to temperature and light stimuli. We propose that GsCPK16-dependent phosphorylation and activation of GsPIP2;2 regulate gentian flower re-opening, with stimulus-induced Ca2+ signals acting as triggers., (� American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)